Humoral immunity against the proline-rich peptide epitope of the IgA1 hinge region in IgA nephropathy

The study was performed to investigate the role of the IgA1 hinge region in the IgA1-IgA1 interaction, which was observed previously in IgA nephropathy. The competitive inhibition assays of the IgA1-IgA1 binding were performed using the following candidates for inhibitors: native IgA1 hinge glycopeptide (nHGP), IgA1, IgA2, and IgG. The IgA1-IgA1 binding was definitely inhibited by the nHGP and the IgA1 (maximum of percent inhibition: 66.1 and 60.5%, respectively). There was no obvious inhibition in the IgA2 and the IgG. The inhibition curves of the nHGP and the IgA1 were significantly different from that of the IgG (P < 0.01, respectively). Furthermore, to reveal the detailed binding sites in the interaction, the same inhibition assays were performed using the following substances composing the IgA1 hinge glycopeptide: galactose (Gal), N-acetyl-galactosamine (GalNAc), Gal beta 1-3GalNAc, sialic acid, tetrapeptide PTPS, and synthesized hinge proline-rich peptide PVPSTPPTPSPSTPPTPSPS (sHP). sHP, Gal beta 1-3GalNAc, Gal, and GalNAc inhibited the binding (69.3, 34.1, 14.9, 14.6%, respectively). No obvious inhibition was observed in sialic acid and tetrapeptide PTPS. The inhibition curve of sHP was significantly different from that of the PTPS (P < 0.05). Those of Gal beta 1-3GalNAc, Gal, and GalNAc were also significantly different from that of sialic acid (P < 0.05, respectively). These results suggested that the IgA1-IgA1 interaction could be mediated by the core structure including the peptide and the sugars, except for sialic acid in the hinge region, resulting in the formation of the circulating macromolecular IgA1 in IgA nephropathy.

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FP200Comparison of IgA1 hinge-region O-glycoforms between patients with IgA nephropathy and healthy subjects

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfz106.fp200 ◽

2019 ◽

Vol 34 (Supplement_1) ◽

Author(s):

Yukako Ohyama ◽

Kazuo Takahashi ◽

Hisateru Yamaguchi ◽

Shoko Matsushita ◽

Kazuki Nakajima ◽

...

Keyword(s):

Iga Nephropathy ◽

Healthy Subjects ◽

Hinge Region

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P0356QUANTITATIVE ASSESSMENT OF GALNAC NUMBER IN THE HINGE REGION OF IGA1 IN IGA NEPHROPATHY WITH CRESCENTS FORMING

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0356 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Guizhen Yu ◽

Yong Zhang ◽

Xinfang Xie ◽

Bo Meng ◽

Wantao Ying ◽

...

Keyword(s):

Mass Spectrometry ◽

Iga Nephropathy ◽

Validation Cohort ◽

High Resolution Mass Spectrometry ◽

Statistical Significance ◽

Hinge Region ◽

P Value ◽

Healthy Controls ◽

Discovery Cohort ◽

Content Ratio

Abstract Background and Aims Aberrant IgA1 O-linked glycosylation of IgA1 hinge region (HR) has been shown playing an important role in the pathogenesis of IgA nephropathy (IgAN). Serum levels of galactose-deficient IgA1 (Gd-IgA1) was associated with the development and progression in IgAN, however variations in the composition of IgA1 HR glycoforms are unknown. In this study we aim to quantitative assessment of GalNAc number in the hinge region of IgA1 in IgAN with crescents forming using mass spectrometry. Method Plasma polymeric IgA1 (pIgA1) was purified from a discovery cohort including crescentic IgAN (n=11) non-crescentic IgAN (n=10) and healthy controls (HC, n=10) and a validation cohort (crescentic IgAN, n=10; non-crescentic IgAN, n=10 and healthy controls, n=11). After denaturation, reduction, alkylation and trypsin digestion, liquid chromatography-high-resolution mass spectrometry (LC-MS) was used to analyse the IgA1 HR glycans. The intensity of the identified IgA1 O-glycopeptide was calculated and expressed as the relative abundance for each glycopeptide. The molecular weight (MW) of intact O-glycopeptide was calculated as the formula following: MW = HR + x GalNAc + y Gal + z NeuAc + H+ (x, y and z represent the number of GalNAc, galactose and NeuAc which bind to one HR in IgA1 O-linked glycopeptides respectively) Glycopeptide content ratio = (glycopeptide peak intensity/ total glycopeptide intensity) ×100%. Results In the discovery cohort population, the level of Gd-IgA1 was highest in patients with crescentic IgAN, middle in patients with non-crescentic IgAN and lowest in healthy controls (347.69±57.89 U/ml vs 336.32±38.44 U/ml vs 330.14±33.22 U/ml), albeit that didn’t reach the statistical significance. There were significantly difference in GalNAc number of IgA1 HR among patients with IgAN and healthy controls. Overall the numbers of GalNAc bound to one HR was much lower in the patients than healthy controls. As shown in the Figure 1, the proportion of GalNAc 3, defined as O-glycans that were bound to one HR at 3 sites, were highest in patients with crescentic IgAN, then the non-crescentic IgAN and lowest in the healthy controls (9.92%±3.37% vs 6.65%±1.53% vs 4.05%±1.24%; p-value for the trend<0.001). Similar results were observed in GalNAc 4 (45.91%±4.75% vs 41.13%±2.95% vs 40.98%±2.95%; P=0.004. However regarding the GalNAc 5 and GalNAc 6, crescentic IgAN was lowest, then non-crescentic IgAN and highest in the healthy control (GalNAc 5: 45.17%±5.46% vs 46.90%±2.78% vs 48.05%±3.02%, P=0.001 GalNAc 6:1.62%±1.60% vs 3.95%±1.92% vs 5.39%±2.38%; P<0.001). These results were consistent in the validation cohort (Figure 1). Conclusion GalNAc numbers in IgA1 HR was lower in patients with IgAN especially in crescentic IgAN. These results suggest that the O-glycans of IgA1 were associated with the severe phenotype in IgA nephropathy.

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